The replication of (microbial and) eukaryotic genomes Flashcards
High Fidelity of DNA replication
Only about 1 mistake in every 10^10 nucleotides copied
Amazing accuracy – much higher than expected from
accuracy of complementary base-pairing
Error in DNA replication
Sometimes errors can occur
• With small changes in geometry, two hydrogen
bonds can form between G and T
• Rare tautomeric forms of DNA bases occur transiently causing incorrect pairing
Tautomeric Forms
Rare tautomeric forms of DNA bases occur transiently causing incorrect pairing
In the rare tautomeric form, C can pair with A instead of G, etc.
With small changes in geometry, two hydrogen
bonds can form between G and T
DNA ploymerase to rescue
High fidelity of DNA replication important
in initial base-pairing
• Correct nucleotide has a higher affinity
for polymerase (more energetically favorable)
DNA polymerase before covalent binding
• After nucleotide binding before covalent addition,
enzyme must undergo conformational change
where its fingers tighten around the active site
• Occurs more readily with correct base-pairing
• Allows polymerase to double check
DNA polymerase after covalent binding
• The next error-correcting reaction is exonucleolytic
proofreading
• DNA molecules with mismatched nucleotide at 3’
OH end not effective
DNA polymerase self corrects
• 3’-to-5’ proofreading exonuclease clips off
any unpaired residues at the primer
• DNA polymerase functions as a self correcting enzyme with 5’–3 ’ DNA
synthesis activity and 3’–5’ exonuclease
activity
A need for proofreading may explain the 5’ to 3’ direction of DNA chain growth
Growth in 5’-to-3’ direction allows chain to continue to
be elongated when a mistake has been removed
by exonucleolyticm proofreading
Strand-directed Mismatch Repair in prokaryotes
In E.coli, DNA methylation adds methyl groups to all A
nucleotides in the sequence GATC, but not immediately during replication
• Therefore, GATC sequences that have not yet been
methylated are in the new strands just behind the
replication fork
• Three step process
– Recognition of a mismatch
– Excision of the segment of DNA with mismatch
– Resynthesis of the excised segment using old strand as template
• Reduces number of errors made by factor of 100-1,000
Strand distinction in Eukaryotes
• In eukaryotes, mechanism for distinguishing newly
synthesized strand from parental template does not
depend on DNA methylation
• Newly synthesized lagging-strand transiently contains nicks which provides the signal that directs the mismatch proof reading system
• However, this also requires the newly synthesized DNA on the leading strand to be transiently nicked – how this occurs is uncertain
Model for strand-directed mismatch repair in eukaryotes
• MutS binds specifically to mismatched base
pair
• MutL scans nearby DNA for a nick, triggers
degradation of nicked strand all the way back
through the mismatch
Medical implications of mismatch repair
In humans seen in people who inherit one
defective copy of a mismatch repair gene
• Marked pre-disposition for certain cancers like
hereditary nonpolyposis colon cancer
• Spontaneous mutation of remaining
functional gene produces clone of somatic
cells that accumulate mutations very rapidly
DNA organisation in eukaryotes
• Chromosomes (found in the nucleus) – DNA content and complexity – DNA packaging – Inheritance • Extranuclear (extrachromosomal) DNA – Organelle genomes – Coding capacity – Inheritance
Eukaryotic chromosomes
• DNA is organised as fibre like-structures called
chromosomes
• Each chromosome consists of a single linear
DNA molecule
– No. of chromosomes varies between different
species
• Eukaryotic organisms generally diploid (two
copies of each chromosome)
– Prokaryotes are haploid (one copy per cell)
Chromosome structure: chromatin
DNA must be highly compacted to fit into the cell
• Nucleosome is composed of
– A core region comprising two copies each of the histone proteins H2A, H2B, H3 and H4
• Histones are small basic proteins (positive charge, rich in Lys and Arg)
– DNA wound around the core
• 146 bp of DNA/octomer of histones plus ~50 bp linker DNA
• Wrapped as slightly less than 2 turns around the core
• An associated histone H1 protein binds the linker DNA and is involved in compaction
Nucleosome
The nucleosome is the basic structure of chromatin
– invariant component of euchromatin and heterochromatin
Nucleosome structure
• Linker DNA connects nucleosomes – Resembles “beads on a string” – ~50 bp • Nucleosomes = beads • Linker DNA = string
Centromeres and Telomeres
- Centromeres situated near the centre
* Telomeres situated at the ends
Chromosome structure: centromeres
One centromere per chromosome
• Directs chromosome segregation during mitosis and
meiosis
– Contains site at which sister chromatids are paired before segregation
– Section that pulls chromosomes to either pole
– Interacts with proteins (kinetochore) and microtubules
• Contains short, highly-repetitive sequences (satellite DNA)
Mitosis
• Mitosis is the process where replicated chromosomes partition equally into the two daughter cells
– Comes after chromosome has been replicated to give sister chromatids
– These segregate into the two daughter cells to give
identical genetic content
• Generates new cells for the growth and maintenance
of the organism
• Mitosis maintains chromosome number
phase of mitosis
• Replication of chromosomes to give sister chromatids
happens before mitosis (S phase)
• Prophase
– Nuclear membrane breaks down
• Metaphase
– Chromosomes align along equator of cell
• Anaphase
– Chromatids separate and move towards the poles
• Telophase
– Nuclear membranes reform, cell splits in two
Problem replicating the end of chromosomes
• Problem replicating end of chromosomes in eukaryotes
– Telomeres
– Eukaryotic chromosomes linear not circular
• Leading strand can proceed to end
• Lagging strand
– 3’ overhang
– Daughter shorter and continue to get shorter
every time replicate
Telomeres cap the end of eukaryotic chromosomes
• Telomeres form protective cap at ends of chromosomes
• A protein complex called shelterin sequesters the 3’
overhang
– Prevents the 3’ overhand from being mistaken as double strand break and dealt with by DNA repair machinery
• Put multiple copies of non-coding tandem repeat
sequence at ends
• Nonsense DNA later added back after replication by
telomerase
How does telomere DNA replicate?
- The 3’ end of the parental DNA strand is extended by RNA templated DNA synthesis
- Replication of the lagging strand at the chromosome end can be completed by DNA polymerase, using these extensions as a template
Telomerase
• Telomere DNA sequences are recognized by sequence-specific
DNA binding proteins that attract an enzyme called
telomerase
• Telomerase recognizes tip of existing telomere DNA repeat sequence and elongates it in the 5’ to 3’ direction using an RNA template that is a component of the enzyme itself
• The enzymatic portion of telomerase has a reverse
transcriptase activity, i.e. synthesizes DNA using an RNA template
• This extends the parental DNA strand so that replication of the lagging strand at the chromosome end can be completed by DNA polymerase
Telomere length as a measuring stick
• Without telomerase activity, cells can lose 100-200
nucleotides from each telomere every division
• After many generations, the descendent cells will inherit defective chromosomes and ultimately cease dividing
–replicative cell senescence
– counting mechanism in somatic cells
• This safeguards against uncontrolled cell proliferation
Werner syndrome is a premature aging
disease
• Begins in adolescence or early adulthood
• Results in old appearance by 30-40 years
• Physical characteristics:
– Short stature (common from childhood on)
– Bird-like features
– Baldness, cataracts, muscular atrophy
• Inherited, autosomal, recessive trait
• Cells from WS patients have shorter
telomeres
• Mutation in WRN gene (codes for the
helicase part of Telomeric cap structure),
which promotes telomere instability
